A novel microfluidic platform to study exosome biology in PAH.

一种用于研究多环芳烃外泌体生物学的新型微流体平台。

• 批准号: 10158068
• 负责人: VINICIO A DE JESUS PEREZ
• 金额: $ 23.64万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-25 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10158068
• 关键词: Acoustics; Address; Animal Model; Antibodies; Bioinformatics; Biological; Biological Markers; Biology; Blood Vessels; CD81 gene; Cardiovascular system; Cell Culture Techniques; Cells; Characteristics; Chronic; Culture Media; Cultured Cells; Data Set; Disease; Endothelial Cells; Endothelium; Etiology; Experimental Models; Exposure to; Genetic; Goals; Heart failure; Inflammation; Inflammatory; Injury; Lab-On-A-Chips; Life; Lung; Machine Learning; Mediating; Methods; Microfluidics; Molecular; Molecular Genetics; Nucleic Acids; Outcome; Pathologic; Pathologic Processes; Pattern; Phase; Phenotype; Physiological; Physiological Processes; Population; Process; Production; Proteomics; Reporting; Reproducibility; Resolution; Role; Seeds; Signal Transduction; Sorting - Cell Movement; Stress; Surface; Techniques; Technology; Testing; Therapeutic Agents; Time; Vascular remodeling; Vision; angiogenesis; base; cytokine; design; endothelial dysfunction; exosome; extracellular vesicles; hemodynamics; machine learning algorithm; magnetic beads; monolayer; multiple omics; new technology; novel; novel therapeutics; pressure; protein metabolite; pulmonary arterial hypertension; response; shear stress; stressor; technological innovation; tool; transcriptome sequencing

The endothelium is the cellular monolayer that covers the inner lining of the entire circulatory system. Endothelial dysfunction is a feature of pulmonary arterial hypertension (PAH), a life-threatening disease associated with abnormally high pulmonary pressures and chronic right heart failure. Due to the limitations of available static cell culture and animal models, our understanding of the mechanisms that orchestrate the initiation and perseverance of endothelial dysfunction in PAH remains incomplete. Given that endothelial dysfunction is a common finding in PAH, an understanding of the mechanism behind maladaptive endothelial responses could help accelerate the discovery of novel therapies for PAH. Presently, it is believed that endothelial derived exosomes contribute to PAH by carrying signals that trigger maladaptive endothelial responses in the setting of injury. Exosomes are cell-derived small (~30-150 nm) extracellular vesicles that carry proteins, metabolites and nucleic acids involved in a variety of physiological and pathological processes. While it is known that exosomes carry molecular and genetic factors associated with angiogenesis, inflammation and vasoreactivity, a comprehensive assessment of exosome cargo of healthy and dysfunctional PMVECs has been hindered by current low-yield exosome isolation techniques. These techniques cannot perform real-time dynamic exosome isolation from pulmonary microvascular endothelial cells (PMVECs) exposed to PAH-associated stressors. To address this unmet need, we have designed the MFES (Multifunctional Exosome Sorter) that can dissect the whole exosome population into subpopulations based on size and surface markers. MFES is the first lab-on-a-chip platform that integrates: 1) a vessel-on-a-chip module for real-time characterization of PMVEC functional responses across a wide range of physiological and pathological parameters, 2) a module for high-yield exosome size-based isolation, 3) a surface marker based exosome sorting using magnetic beads, and 4) multi-omics phenotyping of exosomes of PMVECs. Here, we are proposing a technology that can enable broadly to investigate the two main defining characteristics of exosomal subtypes, i.e., size and surface markers, both separately independently, and in combination sequentially. We will characterize changes in exosome cargo in healthy and PAH PMVECs exposed to shear stress-related conditions in the MFES. We will isolate subpopulations of exosomes based on size and surface markers and characterize them for their cargo (Aim 1). Then, we will determine whether exosomes derived from stressed PMVECs can induce pathological changes in healthy PMVECs cultured in a microfluidic culture chip (Aim 2). This technological innovation enables to study endothelial exosome biology in a setting that represents the flow dynamics associated with PAH. Further, the use of cutting-edge -omics technologies, bioinformatic analysis integrated with machine learning algorithms to analyze the purified exosomes is expected to yield a comprehensive dataset of exosome cargo profiles and open exciting opportunities for investigating the biological role of exosomes in PAH pathobiology and the testing of novel therapeutic agents.

内皮是覆盖整个循环系统内壁的单层细胞。内皮细胞 功能障碍是肺动脉高压（PAH）的一个特征，这是一种与肺动脉高压相关的危及生命的疾病 异常高的肺动脉压和慢性右心衰竭。由于可用静态单元的限制 文化和动物模型，我们对协调启动和坚持的机制的理解 PAH 中内皮功能障碍的研究仍然不完整。鉴于内皮功能障碍是常见的发现 PAH，了解适应不良内皮反应背后的机制可能有助于加速 发现 PAH 的新疗法。目前，人们认为内皮来源的外泌体有助于 PAH 通过携带信号在损伤时触发适应不良的内皮反应。外泌体是 细胞来源的小（~30-150 nm）细胞外囊泡，携带相关蛋白质、代谢物和核酸 各种生理和病理过程中。虽然已知外泌体携带分子和 与血管生成、炎症和血管反应性相关的遗传因素，综合评估 目前外泌体分离产量低，阻碍了健康和功能失调的 PMVEC 的外泌体运输 技术。这些技术无法从肺部实时动态分离外泌体 微血管内皮细胞 (PMVEC) 暴露于 PAH 相关应激源。为了解决这一未满足的需求， 我们设计了 MFES（多功能外泌体分选机），可以解剖整个外泌体群体 根据大小和表面标记分为亚群。 MFES 是第一个芯片实验室平台，集成了： 1) 片上容器模块，用于实时表征大范围 PMVEC 功能响应 生理和病理参数，2) 用于基于大小的高产外泌体分离的模块，3) 使用磁珠进行基于表面标记的外泌体分选，以及 4) 外泌体的多组学表型分析 PMVEC。在这里，我们提出了一种技术，可以广泛地研究两个主要定义 外泌体亚型的特征，即大小和表面标记，两者分别独立，并且在 依次组合。我们将描述健康和暴露的 PAH PMVEC 中外泌体货物的变化 MFES 中与剪切应力相关的条件。我们将根据大小和大小来分离外泌体的亚群 表面标记并描述其货物特征（目标 1）。然后，我们将确定外泌体是否 源自应激 PMVEC 的细胞可以诱导在微流体中培养的健康 PMVEC 发生病理变化 培养芯片（目标 2）。这项技术创新使得能够在以下环境中研究内皮外泌体生物学： 代表与 PAH 相关的流动动力学。此外，利用尖端组学技术， 预计将生物信息分析与机器学习算法相结合来分析纯化的外泌体 产生外泌体货物概况的综合数据集，并为研究提供令人兴奋的机会 外泌体在 PAH 病理学和新型治疗药物测试中的生物学作用。